Stress of adult bone by mechanical loading stimulates new bone formation. There have been many in vitro models and examples of this phenomenon in vivo studied in recent years. The mechanisms by which mechanical loading stimulate bone formation are unknown, but the basis for stating a cogent testable hypothesis has been developed. A basic mechanism of cell-matrix interaction has been discovered, that of cellular integrins serving as matrix proteins. Integrins are heterodimers of alpha and beta components, with each component having large extracellular domains responsible for ligand binding, a transmembrane domain, and a short cytoplasmic domain responsible for interacting with the actin cytoskeleton and organizing signal transduction. The central hypothesis of this project is that mechanical strain activates specific integrins in osteoblast focal adhesions lead to generation of cell signals that culminate in osteoblast precursor proliferation, increased production of bone matrix proteins, and bone formation. Secondary hypothesis are integrin signaling for proliferation may be determined by the recruitment of the adaptor protein. Shc to the integrin signaling complex; that mechanical loading translocates and activates the non-receptor protein tyrosine kinase, c- src; and that the p21ras pathway involving the rho family of GTP binding proteins are activated loading to stimulation of MAP kinase and downstream transcription factors, immediate early gene activation and eventually to cell cycle progression, replication, differentiation and bone formation. The specific aims are to: 1) characterize the effects of mechanical strain on osteoblast cell-matrix interactions; 2) characterize signal transduction in the osteoblast response to mechanical loading. Studies will be performed in cultures of human osteoblasts and marrow stromal cells differentiating along the osteoblastic program. Blocked antibodies, antisense strategies, and dominant negative transfections will be used to interdict specific pathways identified as key during the studies. As the result of the studies in this proposal, we should develop a careful analysis of how an important anabolic factor, mechanical loading, works to build bone. This should serve as a foundation to analyze manipulations of the pathway for therapeutic purposes. Techniques for stimulating new bone formation are badly needed in modern medicine.